The two most common methods for measuring absolute or relative protein amounts are protein assays and quantitative Western or immuno-blots. All methods for protein quantitation start with the isolation of large quantities of the cell type of interest due to the limited sensitivity and detection capabilities of these techniques, making them time consuming and laborious. The cellular resolution of these techniques is also limited because the isolated tissue is typically a heterogeneous population of cells that can include a wide range of cell types outside of a user's interest. These techniques are necessarily destructive processes as cells must be lysed to extract their protein content to be manipulated for the detection processes. Inaccuracies in quantitation using immuno-detection are further compounded by variabilities in antibody used to detect the protein, such as the avidity and affinity of the antibody, access of the antibody to the protein epitope, phosphorylation state of the protein, and cross-reactivities of the antibody. The use of a “housekeeping” protein for normalization is subject to the same limitations, as housekeeping protein quantification is still dependent on antibody detection, and differences across conditions, along with cellular heterogeneity can increase or decrease the housekeeping protein quantified without affecting the protein of interest (e.g., epithelial cells within neural tissue may not express a neural protein), leading to an inaccurate ratio between the protein of interest and the normalization control.
It would be highly desirable to be provided with a protein quantification method which would have heightened sensitivity and/or sensibility. It would also be desirable to be provided with a non-destructive protein quantification method that could allow, for example, live cell tracking. It would further be desirable to be provided with a protein quantification method that could be applied to a single cell. It would also be desirable to track and quantify protein production amounts over time in a cell by performing real-time measurements of protein production in single cells, at cellular or sub-cellular resolution.